Toxicological Sciences 55, 69-77 (2000)
Copyright © 2000 by the Society of Toxicology
Endocrine Toxicology |
A Rapid and Sensitive Reporter Gene that Uses Green Fluorescent Protein Expression to Detect Chemicals with Estrogenic Activity
* Department of Anatomy and Public Health and
Department of Pathobiology, College of Veterinary Medicine, and
Department of Animal Science, Texas A&M University, College Station, Texas 77843
A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10–9 M 17ß-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000–3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10–12 M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0–10 M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10–7 M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2–4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17ß-estradiol.
Key Words: cellular steroid receptors; endocrine disruption; xenobiotics; transfection; MCF7-ERE cells; estrogenic activity.
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